Project Summary Phenotypically normal tissue, such as the skin, is capable of harboring oncogenic mutations, yet it is unclear how cells are behaving at this interface between homeostasis and cancer and when/how this balance is tipped to either maintain normalcy or result in tumor emergence. A major challenge that has greatly limited our understanding of what goes awry during the emergence of cancer is the inability to follow the same cells over time in an intact animal. Specifically, this roadblock hinders our ability to understand both the role of specific cells and how their location contributes to their growth, be it normal or cancerous. Our lab is uniquely poised to address these questions since we have pioneered the use of live imaging in skin stem cell regeneration and established an approach that allows us to follow the same cells over time in an intact, live mouse. Stem cells and their environment, the so-called niches, are critical components that sustain not only proper tissue homeostasis, but also diseased states such as cancer. Through 2-photon live imaging, we have learned that first, the location where stem cells reside in the lower part of the hair follicle influences their fate. Second, the induction of oncogenic mutations in clones, such as mutations that stabilize the Wnt effector ?-catenin, will drive aberrant growths non-cell autonomously through the recruitment of wild-type cells. Based on this preliminary data, we are now utilizing 2-photon live imaging to capture the cellular behaviors present at the interface between homeostasis and cancer prior to the development of macroscopic phenotypes. To identify the aberrant stem cell/progenitor activities that lead to tumorigenesis, we are utilizing a well- characterized mutation found across a variety of different cancers, including skin cancer- activated Hras. We will then add loss of TGF? signaling to this system, which is established to synergize with activated Hras to result in the production of cutaneous Squamous Cell Carcinoma (cSCC). Within this system, we plan to utilize 2-photon live imaging to characterize how mutational burden, with respect to both number and position, influences a tissue's ability to cope at both the tissue and the cellular levels. We will then correlate these observed cellular behaviors with specific molecular characteristics as well as biochemical signaling pathways. Mutations will be induced at clonal levels throughout the skin epithelium to enable our observation of how mutant clones interface with homeostatic tissue. To generate these mutations in mice, a combination of genetic and viral tools, including in utero viral injection, will be utilized. Through studying the interface between homeostasis and cancer, we hope to elucidate mechanisms that can later be applied not only towards the treatment of cSCCs, but of any tumors that share comparable genetic and pathophysiological mechanisms.